Insulin is secreted from the pancreatic islet beta cells in response to elevated blood glucose levels and functions to control systemic metabolism and growth. Dysfunction of glucose-stimulated insulin secretion (GSIS) is a hallmark of both Type I and Type II diabetes. Thus, an understanding of the molecular mechanisms that participate in regulating GSIS is essential to understanding both systemic control of metabolic homeostasis as well as the origin and treatment of diabetic disease states. Our recent work has provided evidence supporting a positive regulatory effect of secreted insulin on GSIS. Insulin treatment of cultured beta cells results in activation of the glucose-sensing enzyme, glucokinase. This process is mediated by production of nitric oxide on secretory granules by nitric oxide synthase. However, the signaling pathway leading to the activation of this enzyme is not understood. In addition, it is unclear whether insulin stimulates a similar auto feedback pathway in the crowd of beta cells and other cell types as occurs in an islet and confers a similar regulatory potential on GSIS through the activation of nitric oxide synthase and glucokinase. Based on our preliminary studies, we hypothesize that insulin activates nitric oxide synthase by stimulating the release of calcium ions from intracellular stores and that this process occurs in pancreatic islets. Therefore, the Specific Aims of this study are to 1) determine the molecular signaling events leading from activated insulin receptors to activation of NOS on secretory granules and [unreadable] 2) determine whether insulin treatment activates nitric oxide synthase and glucokinase in living pancreatic islets. To accomplish these goals, we have developed genetically-encoded biosensors targeted to specific cellular compartments in order to measure insulin signal transduction in living cells. This will allow direct examination of nitric oxide production on individual granules in living beta cells by fluorescence imaging techniques. In addition, a lentiviral vector system will be developed in order to specifically deliver the biosensor constructs to beta cells in isolated pancreatic islets. These studies will further understanding of the physiological regulation of GSIS and may identify novel targets for pharmacological intervention of Type I and Type II diabetes. [unreadable] [unreadable]